Dna dosage forms

ABSTRACT

The present invention relates to DNA formulations suitable for ballistic delivery into the skin of the human body. In particular the present invention provides DNA formulations suitable for ballistic administration of DNA vaccines into the skin. The present invention provides a novel DNA pharmaceutical agent dosage form, having a dense core element which is coated with an amorphous solid reservoir medium containing the DNA pharmaceutical agent.

The present invention relates to DNA pharmaceutical formulations, andpreferably those formulations that are suitable for ballistic deliveryinto the skin of the human body. The present invention provides a novelDNA pharmaceutical agent dosage form, having a dense core element whichis coated with an amorphous solid reservoir medium containing the DNApharmaceutical agent in solid solution or suspension within it. Thedense core element is preferably a small metal bead suitable forballistic delivery of the agent into a cell, commonly such beads areroughly spherical gold or tungsten microbeads of an average particlesize in the range of between 0.5 to 10 micrometers in diameter.Preferably the solid pharmaceutical reservoir medium coating the beadsis a polyol, preferably being a polyol in an amorphous state. Preferablythe polyol is a carbohydrate such as trehalose or sucrose. The solidpharmaceutical reservoir medium may further comprise a stabilising agentthat inhibits the degradative effects of free radicals, such as a freeradical scavenger or a metal ion chelator. The DNA pharmaceuticalformulations of the present invention are storage stable, in that theDNA is stabilised in its supercoiled form, and only substantiallyrelease the DNA after administration to the skin. Furthermore, vaccinedelivery devices for the administration of the DNAvaccines into the skinare provided, methods of their manufacture, and their use in medicine.

The skin represents a significant barrier to external agents. A summaryof human skin is provided in Dorland's Illustrated Medical Dictionary,28^(th) Edition. Starting from the external layers, working inwards, theskin comprises the epithelium consisting of the stratum corneum and theviable epithelium, and underlying the epithelium is the dermis. Theviable epithelium consists of four layers: Stratum comeum, Stratumlucidium, Stratum granulosum, Stratum spinosum, and Stratum basale. Theepithelium (including all five layers) is the outermost non-vascularlayer of the skin, and varies between 0.07 and 0.12 mm thick (70-120μm). The epithelium is populated with keratinocytes, a cell thatproduces keratin and constitutes 95% of the dedicated epidermal cells.The other 5% of cells are melanocytes. The underlying dermis is normallyfound within a range of 0.3 to about 3 mm beneath the surface of thestratum corneum, and contains sweat glands, hair follicles, nerveendings and blood vessels.

The stratum corneum dominates the skin permeability barrier and consistsof a few dozen horny, keratinised epithelium layers. The narrowinterstices between the dead or dying keratinocytes in this region arefilled with crystalline lipid multilamellae. These efficiently seal theinterstices between the skin or body interior and the surroundings byproviding a hydrophobic barrier to entry by hydrophilic molecules. Thestratum corneum being in the range of 30-70 μm thick.

Langerhans cells are found throughout the basal granular layer of theviable epithelium (stratum spinosum and stratum granulosum, (SmallAnimal Dermatology-Third Edition, Muller-Kirk-Scott, Ed: Saunders(1983)) and are considered to play an important role in the immunesystem's initial defence against invading organisms. This layer of theskin therefore represents a suitable target zone for certain types ofvaccine.

Conventional modes for administration of pharmaceutical agents into oracross the skin, most commonly by hypodermic needle and syringe, areassociated with numerous disadvantages. Such disadvantages include pain,the requirement for trained professionals to administer the agent, andalso the risk of needle-stick injuries to the administrator with theaccompanying risk of infection with a blood born disease. As such, thereis a need to improve the method of administration of all types ofpharmaceutical into or through the skin.

A number of alternative approaches have been described in order toovercome the problems of administering agent across the stratum corneum,including various devices for the ballistic delivery of vaccines insupersonic gas flow.

DNA vaccines usually consist of a bacterial plasmid vector into which isinserted a strong viral promoter, the gene of interest which encodes foran antigenic peptide and a polyadenylation/transcriptional terminationsequences. The gene of interest may encode a full protein or simply anantigenic peptide sequence relating to the pathogen, tumour or otheragent which is intended to be protected against. The plasmid can begrown in bacteria, such as for example E.coli and then isolated andprepared in an appropriate medium, depending upon the intended route ofadministration, before being administered to the host. Followingadministration the host cells produce the plasmid encoded protein orpeptide. The plasmid vectors are generally made without an origin ofreplication which is functional in eukaryotic cells, in order to preventplasmid replication in the mammalian host and integration withinchromosomal DNA of the animal concerned. Information in relation to DNAvaccination is provided in Donnelly et al “DNA vaccines” Ann. RevImmunol. 1997 15: 617-648, the disclosure of which is included herein inits entirety by way of reference.

Plasmid based delivery of genes, particularly for immunisation or genetherapy purposes is known. For example, administration of naked DNA byinjection into mouse muscle is outlined in WO90/11092. Johnston et al WO91/07487 describe methods of transferring a gene to veterbrate cells, bythe use of microbeads onto which a polynucleotide encoding a gene ofinterest has been precipitated, and accelerating the DNA/microbeads suchthat they penetrate the target cell. Devices for administration of goldor tungsten beads coated with DNA into cells of the skin are describedin U.S. Pat. No. 5,630,796; WO 96/04947; WO 96/12513; WO 96/20022; WO97/34652; WO 97/48485; WO 99/01168; WO 99/01169. Methods of vaccinationusing crystalline forms of ballistically delivered pharmaceutical agentare described in WO 99/27961. The present invention provides improvedDNA dosage forms for use needleless ballistic delivery devices such asthose described in the above publications. The formulations wherein DNAis precipitated onto gold beads as described in the art have the problemthat it is difficult to co-formulate the DNA with additionalagents/excipients. The present invention provides a method ofco-formulation of additional agents.

Solid dosage forms comprising a pharmaceutical agent (including DNAplasmids) and a stabilising polyol (such as a sugar) wherein the dosageforms are in the form of ballistically delivered powders are describedin WO 96/03978. The stabilisation of agents in amorphous sugar glasseshas been described in U.S. Pat. No. 5,098,893.

Sugars used in pharmaceutical formulations can be either crystalline oramorphous. Amorphous solids are distinguished from crytalline by theirlack of three-dimensional long-range order found in crystallinematerials. Amorphous solids are similar to liquids at a molecular levelwherein the molecules are randomly arranged. Amorphous sugars impartstability to pharmaceutical formulations when stored at temperaturesbelow the glass transition temperature. Amorphous sugars exhibit aproperty in which there is a change in the mobility of the molecules inthe sugar matrix below a temperature called the glass transitiontemperature. Below this temperature (Tg), amorphous sugars exist in aglassy state and above this temperature in a rubbery state. Attemperatures below Tg the mobility of the sugar molecules and anymolecules associated or trapped in the sugar matrix is extremely lowgiving rise to long-term stability of such formulations. In other words,the formation of a glass dramatically reduces the diffusional rates ofthe molecules. This is also accompanied by a decrease in the heatcapacity at constant pressure (Cp) by 40 to 100%. This transition can bereadily observed by sensitive thermal techniques like differentialscanning calorimetry (Duddu, S. P., Zhang G, and Dal Monte, P. R.,1997., Pharm Res., 14: 596-600). The stabilizing properties of sugarshave also been attributed to their hydrogen bonding properties withbiological molecules like proteins.

It is desirable for DNA pharmaceutical agents to be delivered in asupercoiled form. Supercoiled DNA in liquid pharmaceutical preparationsare known to degrade over time resulting in the loss of the supercoiledstructure and associated formation of open circle or linear DNAstructures (Evans et al., 2000, Journal of Pharmaceutical Sciences,89(1), 76-87; WO 97/40839). One mechanism by which this chain scissionreaction may occur is oxidation of the DNA by free hydroxyl radicalsproduced from dissolved oxygen in the DNA solutions, a process that iscatalysed by metal ions. The free radical formation reaction may becatalysed by several transition metal ions, the most common of which,however, are iron and copper ions (Fe⁺³, Fe⁺², Cu⁺² or C⁺¹; Evans et al.spra).

The instability of supercoiled DNA is apparent when the DNA is in liquidsolution. However, removal of trace metal ions from supercoiled DNAcontaining liquid solutions with metal ion chelators, and/or mopping upfree radicals in solution by non-reducing free radical scavengersstabilises the DNA in the supercoiled form and protects the DNA fromoxidation (WO 97/40839). The problem of stabilisation of dry forms ofDNA once coated onto a gold or tungsten bead has hitherto not beenaddressed in the art. Surprisingly, the present inventors have observedthat dry forms of DNA, without the technology of the present invention,when coated onto gold or tungsten microbeads are also unstable.

The present invention overcomes these problems and provides a DNAdelivery dose which is capable of administering and releasing the DNAagents efficiently into the skin, with or without additional excipients,and also in which the DNA is stabilised in its supercoiled form.

DESCRIPTION OF FIGURES

FIG. 1, pVAC1.ova

FIG. 2 shows a graphical plot of the percentage of supercoiled plasmid,(% ccc), both monomeric, (% cccmon), and dimeric, (% cccdim), plasmidforms; after coating and lyophilization onto sowing needles and storageat 37° C. The plasmid formulations used contain varying amounts ofsugars: FIG. 2A: 5% Sucrose, FIG. 2B: 10% Sucrose, FIG. 2C: 17.5%Sucrose, FIG. 2D: 40% Sucrose, FIG. 2E: 40% Trehalose, FIG. 2F: 40%Glucose.

FIG. 3 shows differential scanning calorimetry, (DSC), data, for plasmidDNA, (10 mg/ml), formulations in 40% sucrose. FIG. 3A & B: formulationsalso contain: 100 mM TrisHCl pH8.0, 1 mM EDTA, 10 mM methionine and 2.9%ethanol; FIG. 3A & C represent a 24 hour lyophilization cycle; FIG. 3B &D represent a 1 hour lyophilization cycle.

FIG. 4 shows polarized light microscopy data, for plasmid DNA, (10mg/ml), formulations in 40% sucrose. FIG. 4A: formulations also contain:100 mM TrisHCl pH8.0, 1 mM EDTA, 10 mM methionine and 2.9% ethanol, FIG.4C: only contains 40% sucrose and FIG. 4D: shows crystals of theexcipients described in the formulation shown in FIG. 4A. 1AM, 2AM & 3AMrepresent a 24 hour lyophilization cycle, whereas 1ST, 2ST & 3STrepresent a 1 hour lyophilization cycle.

FIG. 5 shows polarized light microscopy data, for lyophilisized plasmidDNA, (10 mg/ml), formulations in sugars and polyols, which also contain:100 mM TrisHCl pH8.0, 1 mM EDTA, 10 mM methionine and 2.9% ethanol. FIG.5A, sample 1:40% w/v ficoll, sample 2:20% w/v dextran, sample 3:40% w/vsucrose, sample 4:20% w/v maltotriose. FIG. 5B, sample 5:20% w/vlactose, sample 6:30% w/v maltose, sample 7:40% w/v glucose, sample8:40% w/v trehalose.

FIG. 6 shows the stability of supercoiled DNA plasmid coated onto goldbeads and stored for 1 week at 25° C.

FIG. 7 shows the stability of supercoiled DNA plasmid coated onto goldbeads and stored for 3 weeks at 25° C.

The present invention provides a novel DNA pharmaceutical agent dosageform, having a dense core element which is coated with a solid reservoirmedium containing the DNA pharmaceutical agent.

DNA vaccine dosage forms are a preferred aspect of the presentinvention. In such applications the agent to be delivered is apolynucleotide that encodes an antigen or antigens derivable from apathogen such as micro-organisms or viruses, or may be a self antigen inthe case of a cancer vaccine or other self antigen.

The DNA component of the present invention may be linear or opencircular or supercoiled plasmid DNA, but may in a related form of thepresent invention the DNA may be in the form of a live attenuatedbacterial or viral vector.

Certain embodiments of the device described herein also have thesignificant advantage of being stored at room temperature thus reducinglogistic costs and releasing valuable refrigerator space for otherproducts.

The solid amorphous reservoir medium is preferably a polyol that fulfilsthe function required for the present invention. The reservoir must becapable of adhering to the microbead to a sufficient extent that thereservoir remains physically stable and attached during prolongedstorage, and also remains substantially intact during the administrationprocedure when the coated microbead is projected through the stratumcorneum. The reservoir must also be capable of holding or containing asuspension or solution of agent to be delivered in any dry or partiallydry form, which is released into the skin during biodegradation of thereservoir medium.

Biodegradation of the medium in the sense of the present invention meansthat the reservoir medium changes state, such that changes from itsnon-releasing to its releasing states whereby the agent enters into theskin. The release of the active agent may involve one or more physicaland/or chemical processes such as hydration, diffusion, phasetransition, crystallisation, dissolution, enzymatic reaction and/orchemical reaction. Depending on the choice of reservoir medium,biodegradation can be induced by one or more of the following: water,body fluids, humidity, body temperature, enzymes, catalysts and/orreactants. The change of the reservoir medium may therefore be inducedby hydration, and warming associated with the higher humidity andtemperature of the skin. The reservoir medium may then degrade bydissolution and/or swelling and/or change phase (crystalline oramorphous), thereby disintegrating or merely increase the permeation ofthe medium.

Preferably the medium dissolves, and is metabolised or expelled orexcreted from the body, but the reservoir may alternatively remainattached to microbead which may be expelled from the body by severalmechanisms including sloughing off of dead skin cells during normal skinreplacement. Release of the agent by dissolution of the reservoir mediumis preferred.

Preferably the solid reservoir medium is a polyol (such as thosedescribed in WO 96/03978). Suitable polyol reservoir media includecarbohydrates (such as sugars), polysaccharides, substituted polyolssuch as hydrophobically derivatised carbohydrates, amino acids,biodegradable polymers or co-polymers such as poly(hydroxy acid)s,polyabhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid)s,poly(valeric acid)s, and poly(lactide-co-caprolactone)s, or polylactideco-glycolide.

The solid reservoir may be in an amorphous or crystalline state and mayalso be partially amorphous and partially crystalline. Most preferably,however, all or substantially all of the reservoir is in an amorphousstate. More preferably still is that the amorphous reservoir is in theform of a glass (U.S. Pat. No. 5,098,893). Most preferably the reservoiris a sugar glass. A glass reservoir may have any glass transitiontemperature, but preferably it has a glass transition temperature thatboth stabilises the pharmaceutical agent during storage and alsofacilitates rapid release of the agent after insertion of the reservoirinto the skin. Accordingly, the glass transition temperature is greaterthan 30-40° C., but most preferably is around body temperature (such as,but not limited to 40-50° C.).

Particularly preferred reservoir media are those that stabilise theagent to be delivered over the period of storage. For example, antigenor agent dissolved or dispersed in a polyol glass or simply dried in apolyol are storage stable over prolonged periods of time (U.S. Pat. No.5,098,893, U.S. Pat. No. 6,071,428; WO 98/16205; WO 96/05809; WO96/03978; US 4,891,319; U.S. Pat. No. 5,621,094; WO 96/33744). Suchpolyols form the preferred set of reservoir media.

Preferred polyols include sugars, including mono, di, tri, or oligosaccharides and their corresponding sugar alcohols. Suitable sugars foruse in the present invention are well known in the art and include,trehalose, sucrose, lactose, fructose, galactose, glucose, mannose,maltulose, iso-maltulose and lactulose, maltose, or dextrose and sugaralcohols of the aforementioned such as mannitol, lactitol and maltitol.Sucrose, Glucose, Lactose, Raffinose and Trehalose are preferred.

The reservoir mediums of the present invention may preferably furthercontain a stabilising agent that inhibits the degradative effects offree radicals. Preferred stabilising agents include stabilising metalion chelating agents, such preferred metal ion chelating agents includeinositol hexaphosphate, tripolyphosphate, succinic and malic acid,ethylenediamine tetraacetic acid (EDTA), tromethamine (TRIS), Desferal,diethylenetriaminepentaacetic acid (DTPA) andethylenediamindihydroxyphenylacetic acid (EDDHA). Other preferredstabilising agents are non-reducing free radical scavengers, andpreferably such as agents are ethanol, methionine or glutathione. Othersuitable chelators and scavengers (and those which are not suitable) maybe readily identified by the man skilled in the art by routineexperimentation (as described in WO 97/40839).

The preferred solid reservoir media in the devices of the presentinvention contain a metal ion chelating agent or a non-reducing freeradical scavenger. Most preferably the solid reservoir media in thedevices of the present invention contain both a metal ion chelatingagent and a non-reducing free radical scavenger.

The amounts of the stabilising agents may be determined by the manskilled in the art, but generally are in the range of 0.1-10 mM for themetal ion chelators, Ethanol is present in an amount up to about 5%(v/v), methionine is present at about 0.1 to 100 mM and Glutathione ispresent at about 0.1 to 10% (v/v).

Preferred combinations of stabilising agents are (a) Phosphate bufferedethanol solution in combination with methionine or EDTA, (b) Trisbuffered EDTA in combination with methionine or ethanol (or combinationsof methionine and ethanol).

Particularly preferred formulations which may be combined with DNA andcoated onto the dense core elements to form solid dosage forms of thepresent invention contain polyols (preferably sucrose or trehalose)dissolved in demetalated water or Phosphate or Tris based buffers andfurther comprising either:

A. 10 mM methionine and 2.9% ethanol, or

B. 3.7% ethanol and 1 mM EDTA, or

C. 100 mM Tris, 1 mM EDTA and 10 mM methionine and 2.9% ethanol, or

D. 100 mM Tris, 1 mM EDTA and 10 mM methionine, or

E. 100 mM Tris, 1 mM EDTA and 2.9% ethanol.

In the preferred methods of manufacture of the present invention the DNAis stored and handled in these stabilising agents prior to finalformulation with the sugar.

In addition to these stabilising agents, further steps may be taken toenhance the stability of the DNA in the solid vaccines. For example, theformulations may be made using solutions which themselves weredemetalated before use (for example by using commercially availabledemetalating resin such as Chelex 100 from Biorad) and/or theformulation may be finalised in a high pH (such as pH 8-10).

Preferably the DNA is in the form of a supercoiled plasmid. One majoradvantage of the present invention for these formulations is the factthat the DNA is stabilised so that upon release, it largely remains inits supercoiled form, and preferably in its monomeric supercoiled form.

Plasmid DNA stability can be defined in a number of ways and can be arelative phenomenon determined by the conditions of storage such as pH,humidity and temperature. For storage in the presence of iron ions onthe coated reservoir, preferably >50% of plasmid remains supercoiled,(ccc, covalently closed circular), upon storage for 3 months at 4° C.More preferably, under the storage conditions described, >60% of plasmidremains ccc and more preferably, under these storage conditions, >90% ofplasmid remains ccc for 3 months at 4° C. For coating on to non-metalion based needles or microneedles, the stability of plasmid DNA would bepreferably >60% and more preferably 80% and most preferably >90% cccafter 3 months storage at 4° C. More preferably, under these storageconditions, >90% of plasmid remains ccc for 1 year at 4° C., and morepreferably >90% of plasmid remains ccc for 2 years at 4° C. Mostpreferably the above DNA stability is achieved under these conditionsover the same time periods at 25° C.

The DNA within a the solid reservoir medium (for ease of measurement,when coated onto sewing needles) is preferably stabilised in itssupercoiled (ccc) form during accelerated stability studies, and mostpreferably the DNA is stabilised in its monomeric ccc form. An exampleof an acellarated stability study is where dry coated needles aremaintained at 37° C. for 4 weeks followed by analysis of the DNAstructure over time. In this type of study, preferably greater than 50%of the DNA remains in its ccc form, more preferably greater than 60%remains in its ccc form, more preferably greater than 70% remains in itsccc form, more preferably greater than 80% remains in its ccc form andmost preferably greater than 90% remains in its ccc form. Under theseconditions, and preferred levels of ccc, it is also preferred that theratio of monomeric:dimeric ccc DNA is about 1 (such as within the rangeof 0.8-1.2, or more preferably within the range of 0.9-1.1 and mostpreferably within the range of 0.95-1.5), or greater than 1.

Studies to determine plasmid stability are well known to those skilledin the art and are described in (Evans et al., Supra, WO 97/40839).These include techniques to measure and quantify the percentage ofsupercoiled, ccc, plasmid DNA either by agarose gel electrophoresis,anion exchange HPLC, (Ferreira, G. et al., 1999, Pharm. Pharmacol.Commun., 5, pp57-59), or capillary gel electrophoresis, ( Schmidt etal., 1999, Anal. Biochem., 274, 235-240). The ratio of monomeric:dimericccc can be measured by image intensity analysis after agarose gelelectrophoresis (in the absence of any intercalating agents) and EtBrstaining, using commercially available software such as Labworks 4.0running on a UVP Bioimaging system.

In the context of the present invention the solid reservoir medium coatsthe core elements in a manner that the resultant formulation is suitablefor administration by ballistic delivery devices. Accordingly each coreelement may be fully or partially covered by the reservoir, or aplurality of elements may be trapped within a matrix of solid reservoir.In a related method of producing the dosage forms of the presentinvention, a large quantity of reservoir encompassing a large number ofcore elements may be ground into smaller particles which are suitablefor administration by ballistic delivery devices.

Other suitable excipients which may be included in the formulationinclude buffers, amino acids, phase change inhibitors (‘crystalpoisoners’) which may be added to prevent phase change of the coatingduring procesing or storage or inhibitors to prevent deleteriouschemical reactions during processing or storage such Maillard reactioninhibitors like amino acids.

The solid dosage forms of the present invention are used in ballistictransfection of skin cells using devices that entrain the DNA coatedparticles in a gas flow. The particles pass through the stratum corneumand enter into a cell where the DNA is released and expressed by thehost cell. Alternatively, the particle enters the extracellular spaceand releases the DNA therein. Accordingly, the core elements that aresuitable for use in the present invention are those that are suitablefor this purpose. The core elements impart upon the final dosage formsufficient strength and momentum to pierce the stratum corneum in anygiven ballistic delivery device. It is preferred that the core elementshave sufficient density to impart sufficient momentum to the DNA coatedparticles, suitable dense cores have been found to be gold or tungstenmicrobeads. The size of the core elements is preferably that whichimparts sufficient mass to give the required momentum to the DNA coatedparticles, whilst not being too large such that the skin cells suffertoo much damage. Suitable core element particle sizes are those thatwhen coated form particles of a mean diameter in the range of 0.5 to 100μm, preferably between 1 to 50 μm, more preferably between 1 to 10 μm,and most preferably around 2 μm in diameter.

In general the core elements are roughly spherical, although non-regularforms may be used. Most preferably the core elements are gold ortungsten microbeads.

The present invention claims that an amorphous sugar when present withmetal particles and DNA will impart long-term stability to theformulation. Other excipients like surfactants and buffers may beincluded in the formulation.

Examples of methods for the preparation of such amorphous sugarcontaining formulations include:

1. Freeze-Drying

Mix the solution containing sugar, DNA, gold particles and fill intoglass vials. These vials are partially stoppered and loaded into alyophilizer. The shelf temperature is then reduced to −45C leading tothe product in the vials being frozen. After allowing all the vials tofreeze, the condensor is chilled to sub −60C temperature. Primary dryingis then carried out by raising the shelf temperature to approximately−30C while applying a vacuum of approximately 100 mT. During primarydrying the water from the ice crystals that are formed is sublimated.After the primary drying is complete, the shelf temperature is raised toabove ambient temperature and maximum vacuum is applied. The secondarydrying removes any tightly bound water and dries the powder to achievelong term stability.

2. Spray-Drying

Spray drying is a dehydration process that utilizes heat from a hot gasstream (usually air) to evaporate dispersed droplets created byatomization of a continuous liquid feed. Resulting powder products drywithin a few seconds into fine particles. The feasibility of spraydrying for generating therapeutic protein powders has been amplydemonstrated ((Broadhead, J., Rouan, S. K. E., Hau, I., and Rhodes, C.T. 1994. J. Pharm. Pharmacol. 46: 458467.; Mumenthaler, M., Hsu, C. C.,and Pearlman, R. 1994. Pharm. Res. 11: 12-20)). In such an applicationto our formulation mixtures, the formulated DNA, gold particles andsugar solution will be fed into a spray dryer with a typical inlettemperature in the range of 50 to 150C typically at a flow rate between0.1 and 10 mL/min. The resulting powder is dry and is collected from thecollection chamber.

3. Srayfreeze-Drying

Spray freeze-drying is a process in which the solution containing theDNA, gold particles and sugars is sprayed onto trays containing dry iceor liquid nitrogen. This results in the instantaneous freezing of thedroplets. The trays are then loaded into a lyophilizer and the particlesare then freeze-dried according to the process described above.

Using these techniques each solid DNA delivery dose may be loaded withrelatively high amounts of DNA. The formulations resulting from theabove techniques may be used directly or after milling and sieving intoreservoir medium coated dense core beads.

Preferably the vaccine formulations of the present invention contain DNAthat encodes an antigen or antigenic composition capable of eliciting animmune response against a human pathogen, which antigen or antigeniccomposition is derived from HIV-1, (such as tat, nef, gp120 or gp160),human herpes viruses, such as gD or derivatives thereof or ImmediateEarly protein such as ICP27 from HSV1 or HSV2, cytomegalovirus ((espHuman)(such as gB or derivatives thereof), Rotavirus (includinglive-attenuated viruses), Epstein Barr virus (such as gp350 orderivatives thereof), Varicella Zoster Virus (such as gpI, II and IE63),or from a hepatitis virus such as hepatitis B virus (for exampleHepatitis B Surface antigen or a derivative thereof), hepatitis A virus,hepatitis C virus and hepatitis E virus, or from other viral pathogens,such as paramyxoviruses: Respiratory Syncytial virus (such as F and Gproteins or derivatives thereof), parainfluenza virus, measles virus,mumps virus, human papilloma viruses (for example HPV6, 11, 16, 18, . .. ), flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borneencephalitis virus, Japanese Encephalitis Virus) or Influenza virus(whole live or inactivated virus, split influenza virus, grown in eggsor MDCK cells, or Vero cells or whole flu virosomes (as described by R.Gluck, Vaccine, 1992, 10, 915-920) or purified or recombinant proteinsthereof, such as HA, NP, NA, or M proteins, or combinations thereof), orderived from bacterial pathogens such as Neisseria spp, including N.gonorrhea and N. meningitidis (for example capsular polysaccharides andconjugates thereof, transferrin-binding proteins, lactoferrin bindingproteins, PilC, adhesins); S. pyogenes (for example M proteins orfragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S.mutans; H. ducreyi; Moraxella spp, including M. catarrhalis, also knownas Branhamella catarrhalis (for example high and low molecular weightadhesins and invasins); Bordetella spp, including B. pertussis (forexample pertactin, pertussis toxin or derivatives thereof, filamenteoushemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B.bronchiseptica; Mycobacterium spp., including M. tuberculosis (forexample ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M.paratuberculosis, M. smegmatis; Legionella spp, including L.pneumophila; Escherichia spp, including enterotoxic E. coli (for examplecolonization factors, heat-labile toxin or derivatives thereof,heat-stable toxin or derivatives thereof), enterohemorragic E. coli,enteropathogenic E. coli (for example shiga toxin-like toxin orderivatives thereof); Vibrio spp, including V. cholera (for examplecholera toxin or derivatives thereof); Shigella spp, including S.sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y.enterocolitica (for example a Yop protein), Y. pestis, Y.pseudotuberculosis; Campylobacter spp, including C. jejuni (for exampletoxins, adhesins and invasins) and C. coli; Salmonella spp, including S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp.,including L. monocytogenes; Helicobacter spp, including H. pylori (forexample urease, catalase, vacuolating toxin); Pseudomonas spp, includingP. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis;Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp.,including C. tetani (for example tetanus toxin and derivative thereof),C. botulinum (for example botulinum toxin and derivative thereof), C.difficile (for example clostridium toxins A or B and derivativesthereof); Bacillus spp., including B. anthracis (for example botulinumtoxin and derivatives thereof); Corynebacterium spp., including C.diphtheriae (for example diphtheria toxin and derivatives thereof);Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA,DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (forexample OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC,DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agentof the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R.rickettsii; Chlamydia spp., including C. trachomatis (for example MOMP,heparin-binding proteins), C. pneumoniae (for example MOMP,heparin-binding proteins), C. psittaci; Leptospira spp., including L.interrogans; Treponema spp., including T. pallidum (for example the rareouter membrane proteins), T. denticola, T. hyodysenteriae; or derivedfrom parasites such as Plasmodium spp., including P. falciparum;Toxoplasma spp., including S. gondii (or example SAG2, SAG3, Tg34);Entamoeba spp., including E. histolytica; Babesia spp., including B.microti; Trypanosoma spp., including T. cruzi; Giardia spp., includingG. lamblia; Leshmania spp., including L. major; Pneumocystis spp.,including P. carinii; Trichomonas spp., including S. vaginalis;Schisostoma spp., including S. mansoni, or derived from yeast such asCandida spp., including C. albicans; Cryptococcus spp., including C.neoformans. Other preferred bacterial vaccines comprise antigens derivedfrom Haemophilus spp., including H. influenzae type B (for example PRPand conjugates thereof), non typeable H. influenzae, for example OMP26,high molecular weight adhesins, P5, P6, protein D and lipoprotein D, andfimbrin and fimbrin derived peptides (U.S. Pat. No. 5,843,464).

In another embodiment of the present invention the DNA dosage formcontains a DNA vaccine in combination with a non-DNA antigen such as aprotein or polysaccharide antigen derived from a pathogen.

One of the advantages of the present invention is the ability toco-formulate the DNA agent together with additional active agents, anability that has been limited with other solid DNA pharmaceuticalagents. For example, the DNA vaccine may further comprise an agent toenhance uptake of the DNA into the cell, an adjuvant or otherimmunostimulant to improve and/or direct the immune response, and mayalso further comprise pharmaceutically acceptable excipient(s).

For example, the solid pharmaceutical reservoir medium may preferablycontain a DNA condensing agent for example spermidine or PEI(polyethyleneimine). Other excipients which may be included in theformulation include buffers, amino acids, phase change inhibitors(‘crystal poisoners’) which may be added to prevent phase change of thecoating during processing or storage or inhibitors to preventdeleterious chemical reactions during processing or storage suchMaillard reaction inhibitors like amino acids.

A preferred additional agent to the co-entrapped within the reservoirmedium with the DNA is a DNAase inhibitor. One example of a DNAaseinhibitor which is preferred is aurinticarboxylic acid (ATA,Glasspool-Malone, J. et al., (2000), Molecular Therapy 2: 140-146).

Vaccines of the present invention, may advantageously also include animmunologically effective adjuvant in solid solution together with theDNA. Alternatively the adjuvant may be associated with separatemicrobeads to the DNA coated microbead. Suitable adjuvants for vaccinesof the present invention comprise those adjuvants that are capable ofenhancing the antibody responses against the immunogen. Suitableimmunostimulatory agents include, but this list is byno means exhaustiveand does not preclude other agents: synthetic imidazoquinolines such asimiquimod [S-26308, R-837], (Dockrell and Kinghom, 2001, Journal ofAntimicrobial Chemotherapy, 48, 751-755; Harrison, et al. ‘Reduction ofrecurrent HSV disease using imiquimod alone or combined with aglycoprotein vaccine’, Vaccine 19: 1820-1826, (2001)); and resiquimod[S-28463, R-848] (Vasilakos, et al. Adjuvant activites of immuneresponse modifier R-848: Comparison with CpG ODN', Cellular immunology204: 64-74 (2000).), Schiff bases of carbonyls and amines that areconstitutively expressed on antigen presenting cell and T-cell surfaces,such as tucaresol (Rhodes, J. et al. ‘Therapeutic potentiation of theimmune system by costimulatory Schiff-base-forming drugs’, Nature 377:71-75 (1995)), cytokine, chemoline and co-stimulatory molecules aseither protein or peptide, this would include pro-inflammatory cytolinessuch as GM-CSF, IL-1 alpha, IL-1 beta, TGF-alpha and TGF-beta, Th1inducers such as interferon gamma, IL-2, IL-12, I15 and IL-18, Th2inducers such as IL4, IL5, IL-6, IL-10 and IL-13 and other chemokine andco-stimulatory genes such as MCP-1, MIP-1 alpha, MIP-1 beta, RANTES,TCA-3, CD80, CD86 and CD40L, , other immunostimulatory targeting ligandssuch as CTLA-4 and L-selectin, apoptosis stimulating proteins andpeptides such as Fas, (49), synthetic lipid based adjuvants, such asvaxfectin, (Reyes et al., ‘Vaxfectin enhances antigen specific antibodytitres and maintains Th1 type immune responses to plasmid DNAimmunization’, Vaccine 19: 3778-3786) squalene, alpha- tocopherol,polysorbate 80, DOPC and cholesterol, endotoxin, [LPS], Beutler, B.,‘Endotoxin, ‘Toll-like receptor 4, and the afferent limb of innateimmunity’, Current Opinion in Microbiology 3: 23-30 (2000)); CpG oligo-and di-nucleotides, Sato, Y. et al., ‘Immunostimulatory DNA sequencesnecessary for effective intradermal gene inimunization’, Science 273(5273): 352-354 (1996). Hemmi, H. et al., ‘A Toll-like receptorrecognizes bacterial DNA’, Nature 408: 740-745, (2000) and otherpotential ligands that trigger Toll-like receptors to produceTh1-inducing cytokines, such as synthetic Mycobacterial lipoproteins,Mycobacterial protein pl9, peptidoglycan, teichoic acid and lipid A.

Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a Lipid A derivative such asmonophosphoryl lipid A, or preferably 3-de-O-acylated monophosphoryllipid A. MPL® adjuvants are available from Corixa Corporation (Seattle,Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034and 4,912,094). CpG-containing oligonucleotides (in which the CpGdinucleotide is unmethylated) also induce a predominantly Th1 response.Such oligonucleotides are well known and are described, for example, inWO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.Immunostimulatory DNA sequences are also described, for example, by Satoet al., Science 273:352, 1996. Another preferred adjuvant comprises asaponin, such as Quil A, or derivatives thereof, including QS21 and QS7(Aquila Biopharmaceuticals Inc., Framingham, MA); Escin; Digitonin; orGypsophila or Chenopodium quinoa saponins.

In this aspect of the present invention the preferred immunostimulatoryagent or adjuvant is immiquimod or other related molecules (such asresiquimod) as described in PCT patent application publication number WO94/17043 (the contents of which are incorporated herein by reference).

In an embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto small microbeads beads, such as gold, or biodegradablebeads, which are efficiently transported into the cells; or by usingother well known transfection facilitating agents, such as CalciumPhosphate or DEAE dextran.

The amount of expressible DNA in each vaccine administration is selectedas an amount which induces an immunoprotective response withoutsignificant adverse side effects in typical vaccinees. Such amount willvary depending upon which specific DNA construct is employed, however,it is expected that each dose will generally comprise 1-1000 μg of DNA,preferably 1-500 μg, more preferably 1-100 μg, of which 1 to 50 μg isthe most preferable range. An optimal amount for a particular vaccinecan be ascertained by standard studies involving observation ofappropriate immune responses in subjects. Following an initialvaccination, subjects may receive one or several booster immunisationsadequately spaced.

Also provided by the present invention are ballistic delivery devicesloaded with the DNA dosage forms of the present invention.

The formulations of the present invention may be used for bothprophylactic and therapeutic purposes. Accordingly, the presentinvention provides for a method of treating a mammal susceptible to orsuffering from an infectious disease or cancer, or allergy, orautoimmune disease. In a further aspect of the present invention thereis provided a vaccine as herein described for use in medicine. Vaccinepreparation is generally described in New Trends and Developments inVaccines, edited by Voller et al., University Park Press, Baltimore,Md., U.S.A. 1978.

The present invention is exemplified by, but not limited to, thefollowing examples.

EXAMPLE 1 Demonstration of Coating of Microbeads with a Reservoir MediumComprising Plasmid DNA.

Plasmid Preparation and Formulations.

The plasmids used in this study are all shown in FIG. 1. pEGFP-C1 is aGFP expression vector, (Clontech, Palo Alto, Calif., USA). pGL3CMV-is aluciferase expression vector based upon pGL3 Basic, (PromegaCorporation., Madison, Wis., USA), where the CMV immediate earlypromoter drives luciferase expression. pVAC1.ova is a chicken ovalbuminexpression plasmid, constructed by ligating PCR amplified cDNA encodingchicken ovalbumin from pUGOVA, into the expression vector pVAC1. pVAC1is a modification of the mammalian expression vector, pCI, (Promega),where the multiple cloning site, from EcoRI to Bst ZI, has been replacedby the EMCV IRES sequence flanked 5′ by unique Nhe I, RsrII and Xho Iand 3′ by unique Pac I, Asc I and Not I restriction enzyme sites,amplified from pGL3Basic, (Promega). Supercoiled plasmid DNA, (lowendotoxin), was purified on a large scale, aproximately 10 mg yield, tohigh purity using a combination of alkaline SDS lysis, ultrafiltrationand anion exchange column chromatography.

Plasmids were resuspended in TE, (10 mM TrisHCl, 1 mM EDTA), pH 8.0 at 1ug/ul. And determined as >95% supercoiled upon analysis by agarose gelelectrophoresis.

Plasmids were formulated in a variety of solutions, for coating needles,by a standard large-scale ethanol precipitation procedure. Theprecipitated DNA was resuspended directly into the aqueous formulationsolutions at concentrations of 0.5 to 12 ug/ul, (See Chapter 1,Molecular Cloning: A Laboratory Manual, Sambrook, J. et al., 2^(nd)Edition, 1989, CSH laboratory Press, Cold Spring Harbor, N.Y., USA).

1.2 Freeze-Drying

Mix the solution containing sugar (between 1-40% sucrose or trehalose),DNA plasmid, gold particles and fill into glass vials. These vials arepartially stoppered and loaded into a lyophilizer. The shelf temperatureis then reduced to −45C. leading to the product in the vials beingfrozen. After allowing all the vials to freeze, the condensor is chilledto sub −60C. temperature. Primary drying is then carried out by raisingthe shelf temperature to approximately −30C. while applying a vacuum ofapproximately 100 mT. During primary drying the water from the icecrystals that are formed is sublimated. After the primary drying iscomplete, the shelf temperature is raised to above ambient temperatureand maximum vacuum is applied. The secondary drying removes any tightlybound water and dries the powder to achieve long term stability.

1.3 Spray-Drying

Spray drying is a dehydration process that utilizes heat from a hot gasstream (usually air) to evaporate dispersed droplets created byatomization of a continuous liquid feed. Resulting powder products drywithin a few seconds into fine particles. The feasibility of spraydrying for generating therapeutic protein powders has been amplydemonstrated ((Broadhead, J., Rouan, S. K. E., Hau, L, and Rhodes, C. T.1994. J. Pharm. Pharmacol. 46: 458-467.; Mumenthaler, M., Hsu, C. C.,and Pearlman, R. 1994. Pharm. Res. 11: 12-20)). In such an applicationto our formulation mixtures, the formulated DNA, gold particles andsugar solution will be fed into a spray dryer with a typical inlettemperature in the range of 50 to 150C. typically at a flow rate between0.1 and 10 mL/min. The resulting powder is dry and is collected from thecollection chamber.

1.4. Sprayfreeze-Drying

Spray freeze-drying is a process in which the solution containing theDNA, gold particles and sugars is sprayed onto trays containing dry iceor liquid nitrogen. This results in the instantaneous freezing of thedroplets. The trays are then loaded into a lyophilizer and the particlesare then freeze-dried according to the process described above.

The formulations resulting from the above techniques may be useddirectly or after milling in conventional ballistic delivery devices,and expression in target cells may be followed by observing luciferaseexpression. The samples are also stable as measured by maintenance ofsupercoiled structure.

EXAMPLE 2 Stability of Plasmid DNA, in Different Sugar Formulations,After Coating, Lyophilization and Storage on Needles, at 37° C.

A comparison between the plasmid DNA stability of a series of differentDNA formulations was performed where either the amount of sucrose or thetype of sugar used in the formulation was varied. All other excipientspreviously found to be optimal for DNA stability and release werepresent in all formulations, (ie. 100 mM TrisHCl pH8.0, 1 mM EDTA, 10 mMmethionine and 2.9% ethanol). The formulations were compared for theirability to stabilise supercoiled plasmid DNA, after coating andlyophilisation onto needles, upon storage for up to one month at 37° C.,(accelerated DNA stability study). Plasmid DNA (PVAC1.OVA) was theneluted and recovered in the standard manner and subject to agarose gelelectrophoresis, (100V, 100 mA for 2 hours), in the absence ofintercalating agents, (Sambrook, J. et al., supra). The integrity of theeluted plasmid DNA was then monitored after staining with ethidiumbromide and visualisation under UV light. The percentage of supercoiledmonomeric and dimeric plasmid forms and also any linear and opencircular forms from these samples were measured as image intensity usingthe Labworks 4.0 image analysis software on the UVP Bioimaging System.

The data is displayed in FIG. 2, as a graphical plot of the percentageof supercoiled plasmid, (% ccc), both monomeric, (% cccmon), anddimeric, (% cccdim), plasmid forms; after coating and lyophilizationonto sowing needles and storage at 37° C. The plasmid formulations usedcontain varying amounts of sugars: FIG. 2A: 5% sucrose, FIG. 2B: 10%sucrose, FIG. 2C: 17.5% sucrose, FIG. 2D: 40% sucrose, FIG. 2E: 40%trehalose, FIG. 2F: 40% glucose.

The data suggest that all the formulations containing sugars maintain ahigh degree of plasmid stability, even after storage at 37° C. for up toone month, greater than 80% and up to 98% of the plasmid remains in asupercoiled form. For formulations containing sugar levels of 40%,(w/v), the balance between the monomeric and dimeric plasmid formsremains relatively constant, with the preferred monomeric formpredominating in sugar formulations varying from trehalose to sucrose toglucose, (FIGS. 2D, 2E & 2F). For formulations containing lowerconcentrations of sucrose, the dimeric form tends to predominate overthe monomer, especially upon prolonged storage at 37° C., (FIGS. 2A, 2B& 2C). In general the data are consistant with the higher the level ofsugar present in the formulation leading to greater stability of plasmidDNA.

EXAMPLE 3 Demonstration of Amorphous Glass Formation AfterLyophilization of Plasmid DNA, in Sucrose Formulations ContainingExcipients.

Analysis of Lyophilised, Plasmid DNA Formulations by DifferentialScanning Calolimetry, (DSC).

Samples of lyophilised DNA/sucrose formulations were prepared containingplasmid DNA (pVAC1.OVA), (10 mg/ml), in 40% sucrose and also lyophilisedsamples were prepared additionally containing 100 mM TrisHCl pH8.0, 1 mMEDTA, 10 mM methionine and 2.9% ethanol. Samples were split and subjectto either 1 hour or 24 hour lyophilization cycles. The samples were thensubject to analysis by differential scanning calorimetry, (DSC), todetermine the solid state form. This was performed on a TA InstrumentsDSC2920 machine over a temperature range from 25° C. to 300° C., usingnitrogen as the purge gas with a flow rate of 20 ml/min. The sample pantype was pinhole aluninium and the sample weight was determined on theday of analysis on a Mettler M3 balance.

The data is displayed in FIG. 3. All samples contain plasmid DNA, (10mg/ml), in 40% sucrose. FIGS. 3A & B: formulations also contain: 100 mMTrisHCl pH8.0, 1 mM EDTA, 10 mM methionine and 2.9% ethanol; FIGS. 3A &C represent a 24 hour lyophilization cycle; FIGS. 3B & D represent a 1hour lyophilization cycle. The data suggest that all the samples containamorphous sucrose with sucrose glass transition temperatures beingobserved at about 78° C., (FIG. 3A), 85° C., (FIG. 3B), 74° C., (FIG.3C) and 63° C., (FIG. 3D), which fit well with published values in theliterature. The data suggests that both short and long lyophilisationcycles can generate an amorphous sucrose glass. Amorphous glass can formin the presence of high plasmid DNA concentrations and also in thepresence of all the described excipients. However, as it was unclearwhether or not some crystalline material was present in the samples orhad been formed during the DSC analysis itself then further samples wereanalysed by the technique of polarised light microscopy to determine theamorphous/crystalline nature of the samples.

Analysis of Lyophilised, Plasmid DNA Formulations by Polarized LightMicroscopy.

The lyophilised plasmid DNA/sucrose, (±excipients), samples prepared forDSC analysis, described above, were subject to analysis by polarizedlight microscopy. Control samples were prepared of simply 40% sucrose,lyophilised for 1 hour and 24 hour cycles and crystalline samples ofsucrose and the major solid excipients: methionine, Tris HCl and EDTAwere also analysed. This was for comparison and to note the appearanceof any crystalline material present in the formulations. The analysiswas performed on a Zeiss STD16-444111 polarised light microscope withsamples mounted in immersion oil and covered.

The data is shown in FIG. 4 where all formulations contain plasmid DNA,(10 mg/ml), in 40% sucrose. FIG. 4A: formulations also contain: 100 mMTrisHCl pH8.0, 1 mM EDTA, 10 mM methionine and 2.9% ethanol, FIG. 4C:only contains 40% sucrose and FIG. 4D: shows crystals of the major solidexcipient. 1AM, 2AM & 3AM represent a 24 hour lyophilization cycle,whereas 1ST, 2ST & 3ST represent a 1 hour lyophilization cycle.

From FIG. 4C it is clear that both 1 hour and 24 hour lyophilisationcycles performed on 40% sucrose alone generate solely an amorphous glassas expected. From FIG. 4B, the addition of plasmid DNA, (10 mg/ml) tothe 40% sucrose formulation, although it allows largely for theformation of an amorphous glass, does enable the partial formation ofsome crystalline sucrose (2AM and 2ST samples consist of amorphousmaterial with some evidence for some crystal particles, which could besucrose). However, from FIG. 4A, the addition of the excipients to theDNA/sucrose formulation reduces the amount of crystalline particleformulation in samples lyophilised for 24 hours (1AM, the bulk of thesample consists of sheets of amorphous material. There are fewcrystalline particles present), and for short lyophilisation cycles of 1hour, there is no evidence for the formation of crystalline particles,simply an amorphous glass. This suggests that the addition of thedescribed excipients to plasmid DNA in sucrose helps not only to improveDNA release and stability from degradation but also to help preserve theamorphous glass state upon short cycles of lyophilisation.

EXAMPLE 4 Demonstration of Amorphous Glass Formation AfterLyophilization of Plasmid DNA, in Different Sugar/Polyol FormulationsContaining Excipients.

To determine if the nature of the polyol/sugar present in the plasmidDNA formulation with excipients, as described above, affected theability of such formulations to generate an amorphous glass uponlyophilisation, the polyol was varied. A number of similar formulationsthat differed only in the polyol present were generated, lyophilised andanalysed by polarised light microscopy. This was performed in a similarmanner to that described in example 3 except that on this occasion anOlympus BX51 polarized light microscope was used.

The data is shown in FIG. 5 where all formulations contain lyophilisizedplasmid DNA (pVAC1.OVA), (10 mg/ml), and 100 mM TrisHCl pH8.0, 1 mMEDTA, 10 mM methionine and 2.9% ethanol. FIG. 5A, sample 1:40% w/vficoll, sample 2:20% w/v dextran, sample 3:40% w/v sucrose, sample 4:20%w/v maltotriose. FIG. 5B, sample 5:20% w/v lactose, sample 6:30% w/vmaltose, sample 7:40% w/v glucose, sample 8:40% w/v trehalose. Note thatall the samples described and all the formulations analysed formed anamorphous glass with little or no evidence of crystalline material beingpresent. Note that the formulation described as sample 2, containing 20%w/v dextran, was subsequently shown to have precipitated the plasmid DNAout of solution, by agarose gel electrophoretic analysis, (data notshown), and would therefore not be a preferred formulation. This datademonstrates that plasmid DNA plus the described excipients can bemaintained, when lyophilised, in an amorhous glass state by a variety ofpolyols/sugars described in the literature, (Hatley, R. & Blair, J.,(1999), Journal of Molecular Catalysis B: Enzymatic 7: 11-19.).

EXAMPLE 5 Lyophilisation of Sugar/Gold/DNA Formulations

The aim of this study was to lyophilise three sugar based DNAformulations containing gold particles. The formulations were formed asshown below;

-   -   Formulation 1 made up of 40% Sucrose, 100 mM TrisHCl pH8.0, 10        mM L-methionine and 2.9% ethanol    -   Formulation 2 contained 10% Sucrose, 100 mM TrisHCl pH8.0, 10 mM        L-methionine and 2.9% ethanol    -   Formulation 3 was made up of 40% Trehalose, 100 mM TrisHCl        pH8.0, 10 mM L-methionine and 2.9% ethanol

Supercoiled hepatitis B plasmid was formulated into each of the threeformulations at a concentration of 1 mg/mL. Gold particles were added toeach of the formulations at a concentration of 0.5 g per 10 mL offormulation. The formulations were snap frozen by dropwise addition ofeach formulation in liquid nitrogen. The resulting frozen beads weretransferred to 3 mL freeze-drying vials. The vials were freeze dried ina DW8 Heto Holten freeze dryer using the cycle shown below; Stage ofcycle Temperature (° C.)/Vacuum (hPa) Time (hrs) Freezing stage To −40°C. As quickly as possible Hold −40° C. 3 Primary drying −38° C./Vacuum(0.107 hPa) 8 Secondary −38° C. to 5° C./Vacuum (as low as 11 drying(ramp) possible) Secondary  5° C. to 10° C./Vacuum (as low as 2 drying(ramp) possible) Secondary 10° C./Vacuum (as low as possible) 2 drying(hold)

The freeze-dryed samples were stoppered under vacuum. The vacuum wasreleased and the vials removed from the freeze dryer. An acceleratedstability study (at 25° C.) was set up using the freeze-dried DNAformulations. The stability study was monitored at weekly intervalsusing ethidium bromide stained agarose gel electrophoresis.

Agarose Gel Electrophoresis

A 0.6% agarose gel electrophoresis was carried out on stability samplesin order determine a change in DNA conformation during the study. Eachlyophilised sample was reconstituted in distilled water and added to itsrespective well of the agarose gel using the following combination ofsample, loading buffer and distilled water. 2 μl of sample +16 μl ofdistilled water +2 μl of loading buffer

20 μls of each sample was added to the respective well of the gel. Thesamples were electrophoresed overnight at 20 Volts. The electrphoresedgel was stained with ethidium bromide and viewed under UV. The stabilitysamples were assayed following one and three weeks' storage at 25° C.

Results

The photographs of the gels containing the one and three week samplesare shown in FIGS. 6 and 7. FIG. 6: shows the DNA profile of samplesstored at 25° C. for 1 week (lane 1 on left hand side): Lane 1-1ldlobase ladder; Lane 2-Freeze-dried sample (Formulation 1) at 25° C.following one week storage; Lane 3-Freeze-dried sample (Formulation 1)at 5° C. after one week storage-Control; Lane 4-Freeze-dried sample(Formulation 2) at 25° C. following one week storage, Lane5-Freeze-dried sample (Formulation 2) at 5° C. following one weekstorage; Lane 6-Freeze-dried sample (Formulation 3) at 25° C. followingone week storage; Lane 7-Freeze-dried sample (Formulation 3) at 5° C.following one week storage; Lane 8-Pre freeze-dried liquid Formulation 1at 5° C.; Lane 9-Pre freeze-dried liquid Formulation 2 at 5° C. Lane10-Pre freeze-dried liquid Formulation 3 at 5° C.; Lane 11-Unformulatedplasmid, GW700561X (batch A01B30); Lane 12-Freeze-dried Negative controlof Formulation 1 consisting of Formulation 1 diluent+gold beads withoutDNA plasmid

The results showed that

-   -   No significant change in conformation was detected for any of        the three freeze-dried formulations (at 25° C.) when compared to        the controls at 5° C. Most of the DNA was found to be        supercoiled although relatively small amounts of opencircular        and linear topoisoforms were also detected.    -   The pre-freeze dried liquid formulations produced similar band        profiles to the post freeze-dried samples. The higher amount of        fluorescence detected in pre-freeze dried samples is probably        due to the higher concentration of DNA in these.    -   No significant difference in DNA profile detected between the        formulation and unformulated plasmid (lane 11).    -   As expected, no bands were seen in lane 12-the negative control.

FIG. 7 shows the DNA profile of samples stored at 25° C. for 3 week WithLane 1 on the left hand side: Lane 1-1 kilobase ladder; Lane2-Freeze-dried sample (Formulation 1) at 25° C. following three weeksstorage; Lane 3-Freeze-dried sample (Formulation 1) at 5° C. after threeweek storage-Control; Lane 4-Freeze-dried sample (Formulation 2) at 25°C. following three weeks storage; Lane 5-Freeze-dried sample(Formulation 2) at 5° C. following three weeks storage; Lane6-Freeze-dried sample (Formulation 3) at 25° C. following three weeksstorage; Lane 7-Freeze-dried sample (Formulation 3) at 5° C. followingthree weeks storage; Lane 8-Pre freeze-dried liquid Formulation 1 at 5°C.; Lane 9-Pre freeze-dried liquid Formulation 2 at 5° C.; Lane 10-Prefreeze-dried liquid Formulation 3 at 5° C.; Lane 11-Unformulatedplasmid; Lane 12-Freeze-dried Negative control of Formulation 1consisting of Formulation 1 diluent+gold beads without DNA plasmid.

The results showed that

-   -   No significant change in conformation was detected for any of        the three freeze-dried formulations (at 25° C.) when compared to        the controls at 5° C. Most of the DNA was found to be        supercoiled although relatively small amounts of opencircular        and linear isoforms were also detected.    -   The pre-freeze dried liquid formulations produced similar DNA        profiles to the post freeze-dried samples. The higher amount of        fluorescence detected in pre-freeze dried samples is probably        due to the higher concentration of DNA in these.        Conclusions

In this study we have demonstrated that it is possible to freeze drysugar/gold based DNA formulations without a significant change in theDNA conformation. In addition, we have shown that the resultinglyophilised beads were stable at 25° C. for a period of three weeks.

1. A DNA pharmaceutical agent dosage form, having a dense core elementcoated with a solid reservoir medium containing the DNA pharmaceuticalagent.
 2. A DNA pharmaceutical agent dosage form as claimed in claim 1,further comprising a stabilising agent that inhibits the degradativeeffects of free radicals.
 3. A DNA pharmaceutical agent dosage form asclaimed in claim 2 wherein the stabilising agent is one or both of ametal ion chelator and a free radical scavenger.
 4. A DNA pharmaceuticalagent dosage form as claimed in claim 3 wherein the metal ion chelatoris selected from the group consisting of: inositol hexaphosphate;tripolyphosphate; succinic and malic acid; ethylenediamine tetraaceticacid (EDTA); tris (hydroxymethyl) amino methane (TRIS); Desferal;diethylenetriaminepentaacetic acid (DTPA); andethylenediamindihydroxyphenylacetic acid (EDDHA).
 5. A DNApharmaceutical agent dosage form as claimed in claim 3 wherein the freeradical scavenger is selected from the group consisting of ethanol,methionine and glutathione.
 6. A DNA pharmaceutical agent dosage form asclaimed in claim 2 wherein the stabilising agent that inhibits thedegradative effects of free radicals, is a member selected from thegroup consisting of: Phosphate buffered ethanol solution in combinationwith methionine or EDTA; and Tris buffered EDTA in combination withmethionine or ethanol or a combination of methionine and ethanol.
 7. ADNA pharmaceutical agent dosage form as claimed in claim 1, wherein thesolid reservoir medium is an amorphous polyol.
 8. A DNA pharmaceuticalagent dosage form as claimed in claim 7, wherein the polyol is astabilising polyol.
 9. A DNA pharmaceutical agent dosage form as claimedin claim 1 wherein the solid biodegradable reservoir medium is a sugar.10. A DNA pharmaceutical agent dosage form as claimed in claim 9 whereinthe sugar is a member selected from the group consisting of lactose,glucose, sucrose, raffinose and trehalose.
 11. A DNA pharmaceuticalagent dosage form as claimed in claim 1 wherein the solid reservoirmedium is in the form of a glass.
 12. A DNA pharmaceutical agent dosageform as claimed in claim 11, wherein the solid reservoir medium is inthe form of a sugar glass.
 13. A DNA pharmaceutical agent dosage form asclaimed in claim 1, wherein the DNA pharmaceutical agent is supercoiledplasmid DNA.
 14. A DNA pharmaceutical agent dosage form as claimed inclaim 13, wherein the supercoiled plasmid DNA is stabilised such thatafter storage at 37° C. for 4 weeks greater than 50% of the DNA remainsin its supercoiled form.
 15. A DNA pharmaceutical agent dosage form asclaimed in claim 13, wherein the DNA is stabilised such that whenreleased the ratio of monomer:dimer supercoiled form is within the rangeof 0.8:1.2.
 16. A DNA pharmaceutical agent dosage form as claimed inclaim 1, wherein the DNA pharmaceutical agent is a vaccine.
 17. A DNApharmaceutical agent dosage form as claimed in claim 1, wherein thesolid reservoir medium further comprises a member selected from thegroup consisting of vaccine adjuvant, transfection facilitating agent,DNAase inhibitor and a crystal poisoner.
 18. A DNA pharmaceutical agentdosage form as claimed in claim 17, wherein the vaccine adjuvant is amember selected from the group consisting of CpG, a syntheticimidazoquinolines, tucerasol, a cytokines, MPL, QS21, QS7 and an oil inwater emulsions.
 19. A DNA pharmaceutical agent dosage form, as claimedin claim 1 wherein the dense core elements comprises microbeads of amean particle diameter of between 0.5 to 10 μm.
 20. A DNA pharmaceuticalagent dosage form as claimed in claim 19, wherein the microbeads aregold or tungsten microbeads.
 21. A process for the preparation of a DNApharmaceutical agent dosage form as claimed in claim 1, comprisingmaking a solution of DNA pharmaceutical agent, reservoir medium, andstabilising agent that inhibits the degradative effects of free radicalsin an solvent, followed by coating the at least one dense core elementwith said solution, and removing the solvent to form a solid reservoirmedium containing the pharmaceutical agent and agent that inhibits thedegradative effects of free radicals.
 22. A process for the preparationof a DNA pharmaceutical agent dosage form as claimed in claim 21,wherein the reservoir medium is a sugar.
 23. A process for thepreparation of a DNA pharmaceutical agent dosage form as claimed inclaim 22 wherein the concentration of sugar prior to removing thesolvent is in the range of 20-40% w/v.
 24. A process for the preparationof a DNA pharmaceutical agent dosage form as claimed in claim 23,wherein the solvent is demetalated prior to the process.